Synovial CD4+ T cells associate with pain in osteoarthritis: is there a role for fatty acids?

Purpose: Synovitis is associated with pain in patients with knee osteoarthritis (OA). However, the cellular and molecular mechanisms underlying this association remain unclear. Therefore, we characterized the immune cells present in synovium from the knee of OA patients and studied their association with pain. Methods: Infiltrating immune cells were isolated from synovial tissue samples of 40 knee OA patients undergoing total knee-replacement surgery and were characterized by flow cytometry. Perceived pain was determined using visual analogue scale (VAS) for pain. Linear regression was used to determine correlations between the immune cells and pain. Peripheral CD4+ T cells from healthy volunteers were treated with oleic acid in presence or absence of anti-CD3/CD28 and proliferation was measured on day 4. The metabolic state of CD4+ T cells was assessed using Seahorse technology, which measures oxygen consumption rate and extracellular acidification rate in real time. Metabolomics was performed using liquid chromatography/mass spectrometry (LC-MS) and 13C-oleic acid was used to determine 13C-containing lipid species by gas chromatography or LC-MS. Results: Macrophages and CD4+ T cells were most abundant in synovial tissue and both secreted predominantly pro-inflammatory cytokines even without additional stimulation. CD4+ T cells expressed the activation markers CD25 and CD69, indicating an activated state. Linear regression analysis revealed that the percentage of CD4+ T cells, but not macrophages, in synovium was associated with VAS pain (Adjusted B (95% CI)=0.55 (0.09-1.02)), indicating a possible role of these cells in pain perception in knee OA patients. Interestingly, the percentage of CD4+ T cells in synovium also correlated with BMI (r=0.45; p=0.03). Because fatty acid release by joint adipocytes have been previously associated with BMI and because fatty acids have been shown to enhance proliferation of CD4+ T cells, we hypothesized that this could be a relevant mechanism underlying the associations observed in OA synovium. Therefore, we studied the effect of oleic acid on CD4+ T cells. Exposure of oleic acid to CD4+ T cells resulted in a greatly enhanced proliferation. Likewise, when oleic acid was removed after 24h of stimulation an enhanced proliferation was noted, indicating that early oleic acid-induced changes prime CD4+ T cells for enhanced proliferation. To identify these early events, we determined the metabolic state of the cells using the Seahorse technology. While activation of CD4+ T cells led to enhanced glycolysis without affecting mitochondrial respiration during the first 150 min, oleic acid had no effect on either pathway. After 24h of activation similar results were obtained, however a donor-dependent increase in mitochondrial respiration of activated cells was also observed. In line with these data, metabolomics analyses indicated an increase in overall metabolic parameters upon activation of CD4+ T cells for 24h and a modest effect of oleic acid mainly on glycolysis end products (phosphoenolpyruvate) and citric acid cycle begin intermediates (citrate, iso-citrate, cis-aconitate). By using 13C-oleic acid we observed that between 15–40% of oleic acid is taken up from the culture medium during the first 24h and mainly incorporated in phosphatidylcholines and triglycerides, while only 1–2% of oleic acid is metabolized into related fatty acids. Conclusions: Our data indicate that synovial CD4+ T cells are associated with VAS pain in patients with end-stage knee OA, which could represent the cellular basis for the association between synovitis and pain in knee OA patients. This association might be mediated by fatty acids, which greatly enhance proliferation of these cells. Although the underlying mechanisms need further clarification, our studies indicate that oleic acid is likely not used as energy source by CD4+ T cells, but rather incorporated into other lipids which could contribute to the enhanced proliferation of CD4+ T cells.